Improved peptide search for identification of SUMO and sequence-based modifications, in MaxSBM

Post-translational modifications (PTMs), such as SUMOylation and ubiquitination, regulate key cellular processes by covalently attaching to lysine residues. While mass spectrometry allows site-specific identification of PTMs, most existing search engines are optimized for small, non-fragmenting modifications and struggle to detect large, fragmenting protein-based modifiers. We refer to these as Sequence-Based Modifiers (SBMs). To overcome this limitation, we developed an SBM-specific search strategy within MaxQuant that accounts for the fragmentation behavior of SBMs during peptide identification. Using publicly available datasets, we validated our approach for SUMO2/3. Our analysis identified distinct diagnostic features and characteristic mass shifts associated with SBM fragmentation, referred to in this study as d-ions (diagnostic ions) and p-ions. By leveraging these features, our method improved the identification of SUMOylated peptides from human cell lines by 13%, SUMOylation sites in mouse embryonic cells by 18%, and in mouse adipocytes by 25%. Our search method improved spectral annotation of SBMs by up to 9% increase in the median Andromeda score. Taken together, we highlight the potential of our SBM search to enhance the discovery of protein-based modifications.